cells Article Dopamine Receptor Activation Modulates the Integrity of the Perisynaptic Extracellular Matrix at Excitatory Synapses Jessica Mitlöhner 1, Rahul Kaushik 2,3, Hartmut Niekisch 4, Armand Blondiaux 1, Christine E. Gee 5 , Max F. K. Happel 4 , Eckart Gundelfinger 1,3,6 , Alexander Dityatev 2,3,6,*, 1,3, , 1,3,6, Renato Frischknecht * y and Constanze Seidenbecher * 1 Leibniz Institute for Neurobiology (LIN), Department of Neurochemistry and Molecular Biology, 39118 Magdeburg, Germany; [email protected] (J.M.); [email protected] (A.B.); gundelfi@lin-magdeburg.de (E.G.) 2 German Center for Neurodegenerative Diseases (DZNE), Molecular Neuroplasticity Group, 39120 Magdeburg, Germany; [email protected] 3 Center for Behavioral Brain Sciences (CBBS), 39120 Magdeburg, Germany 4 Leibniz Institute for Neurobiology (LIN), Department of Systems Physiology of Learning, 39118 Magdeburg, Germany; [email protected] (H.N.); [email protected] (M.F.K.H.) 5 Center for Molecular Neurobiology Hamburg (ZMNH), Institute for Synaptic Physiology, 20251 Hamburg, Germany; [email protected] 6 Otto-von-Guericke University, Medical Faculty, 39120 Magdeburg, Germany * Correspondence: [email protected] (A.D.); [email protected] (R.F.); [email protected] (C.S.); Tel.: +49-391 67-24526 (A.D.); +49-9131 85-28051 (R.F.); +49-391-6263-92401 (C.S.) Current address: Department of Biology, Animal Physiology, FAU Erlangen-Nürnberg, y 91058 Erlangen, Germany. Received: 12 December 2019; Accepted: 17 January 2020; Published: 21 January 2020 Abstract: In the brain, Hebbian-type and homeostatic forms of plasticity are affected by neuromodulators like dopamine (DA). Modifications of the perisynaptic extracellular matrix (ECM), which control the functions and mobility of synaptic receptors as well as the diffusion of transmitters and neuromodulators in the extracellular space, are crucial for the manifestation of plasticity. Mechanistic links between synaptic activation and ECM modifications are largely unknown. Here, we report that neuromodulation via D1-type DA receptors can induce targeted ECM proteolysis specifically at excitatory synapses of rat cortical neurons via proteases ADAMTS-4 and -5. We showed that receptor activation induces increased proteolysis of brevican (BC) and aggrecan, two major constituents of the adult ECM both in vivo and in vitro. ADAMTS immunoreactivity was detected near synapses, and shRNA-mediated knockdown reduced BC cleavage. We have outlined a molecular scenario of how synaptic activity and neuromodulation are linked to ECM rearrangements via increased cAMP levels, NMDA receptor activation, and intracellular calcium signaling. Keywords: ADAMTS 4/5; brevican; chondroitin sulfate proteoglycan; D1/D5 receptors; NMDA receptors 1. Introduction Synaptic transmission and plasticity are affected by perisynaptic and extrasynaptic factors including the extracellular matrix (ECM), glia-derived components, and neuromodulators. The neuromodulator dopamine (DA) plays an important role in classical and newly discovered forms of synaptic plasticity, such as neo-Hebbian or spike-timing-dependent plasticity (STDP), and hence is fundamental to various forms of learning [1,2]. Dopaminergic modulation of synapses lasts from milliseconds to hours and Cells 2020, 9, 260; doi:10.3390/cells9020260 www.mdpi.com/journal/cells Cells 2020, 9, 260 2 of 21 comprises such diverse mechanisms as regulation of presynaptic neurotransmitter release, e.g., via control of axon terminal excitability or calcium influx, postsynaptic neurotransmitter detection via regulated receptor insertion, or synaptic integration in networks (summarized in Reference [1]). Ultimately, dopaminergic activation also contributes to structural spine plasticity [3]. Dopaminergic signaling is mediated via five different G protein-coupled receptors which can be assigned to two major subgroups: D1-like and D2-like DA receptors [4–6]. Both receptor subgroups have been shown to be coupled to adenylyl cyclase (AC). D1-like receptor activity leads to increased cAMP levels and activation of protein kinase A (PKA) (reviewed in Reference [7]). D1 receptors (D1Rs) are localized both pre- and postsynaptically [8]. D2 (D2R) and D3 receptors have also been found to be expressed both postsynaptically on DA target cells and presynaptically on dopaminergic neurons [8,9]. DA receptors may interact heterologously with other receptor types, such as AMPA-type (AMPARs) and NMDA-type glutamate receptors (NMDARs). This association seems to be important in regulating long-term potentiation (LTP) and working memory [10–12]. NMDARs have been reported to form dynamic surface clusters with D1Rs in the vicinity of glutamatergic synapses. Thus, D1Rs may regulate synaptic plasticity by modulating the synaptic localization of NMDARs [11]. Here, we followed the hypothesis that dopaminergic signaling can also affect the integrity of the hyaluronan (HA)-based extracellular matrix (ECM) that surrounds and stabilizes synapses. This type of ECM structures occurs in the brain as highly condensed perineuronal nets (PNNs) or the more diffuse perisynaptic ECM [13]. The perisynaptic ECM forms a meshwork of macromolecules based on HA as a backbone for chondroitin sulfate proteoglycans like brevican (BC), aggrecan (ACAN), versican or neurocan, glycoproteins, and link proteins (for review, see References [14,15]). As shown by us and others, in-vivo-like ECM structures also develop in dissociated neuronal primary cultures [16–19]. Interestingly, the HA-based neural ECM is formed and remodeled in an activity- and plasticity-dependent manner under in vivo and in vitro conditions [20,21]. Prime candidates for this remodeling are matrix metalloproteases (MMPs) and disintegrin and metalloprotease with thrombospondin motifs (ADAMTS) enzymes [22]. ADAMTS-4 was identified as one of the major proteases processing the proteoglycans ACAN, BC, neurocan, and versican (reviewed in Reference [23]), and is thus a key candidate for ECM remodeling in the brain [24,25]. Controlled cleavage of these proteoglycans results in the generation of defined N- and C-terminal fragments which remain bound to HA-based ECM structures or cell surfaces, respectively (for BC, the fragment sizes are approximately 53 and 80 kDa). ADAMTS enzymes can intrinsically be inhibited by tissue inhibitors of matrix proteases (TIMPs) and activated in response to central nervous system (CNS) injury or disease. However, the molecular pathways mediating neuronal-activity-related control of ADAMTS has remained elusive [26,27]. The extracellular activity of the tissue plasminogen activator (tPA) protease has been shown to be significantly increased in the nucleus accumbens (NAc) of mice after activation of D1-like DA receptors via a PKA-dependent pathway [28]. This enhanced activity is probably associated with an increased release of tPA from vesicles into the extracellular space upon neuronal stimulation, which in turn regulates homeostatic and Hebbian-type synaptic plasticity [28–30]. Here, we hypothesized that proteases modifying the HA-based perisynaptic ECM (i.e., ADAMTS-4 and -5) might also be released or activated after stimulation of D1-like DA receptors. Consequently, we investigated whether activation of DA receptors affects perisynaptic ECM integrity via stimulating the release or activation of ECM-modifying ADAMTS proteases. We found DA receptor agonists that increase cleavage of the ECM constituents BC and ACAN in the rat cortex and in primary cortical cultures, identified the responsible matrix metalloproteases, and unraveled the underlying intracellular signaling mechanisms. Cells 2020, 9, 260 3 of 21 2. Materials and Methods 2.1. Antibodies and Drugs The following primary antibodies were used: rabbit anti-ADAMTS-4 (Abcam, Cambridge, UK), rabbit anti-ADAMTS-5 (OriGene, Rockland, MD, USA), rabbit anti-aggrecan (Merck Millipore, Burlington, MA, USA), rabbit anti-aggrecan neoepitope (Novus Biologicals, Centennial, CO, USA), guinea pig anti-brevican (Seidenbecher et al., 1995) (custom-made; central region of rat BC), mouse anti-brevican (BD Biosciences, San José, CA, USA), rabbit anti-brevican “neo” (Rb399) (custom-made; neo-epitope CGGQEAVESE) [21,31], affinity-purified rabbit anti-brevican “neo” (Rb399) (custom-made; neo-epitope CGGQEAVESE), rat anti-D1 dopamine receptor (Sigma-Aldrich, St. Louis, MO, USA), rabbit anti-D2 dopamine receptor (Abcam, Cambridge, UK), mouse anti-GAD65 (Abcam, Cambridge, UK), rabbit anti-GAPDH (SYSY, Göttingen, Germany), rabbit anti-GFAP (SYSY, Göttingen, Germany), mouse anti- Homer 1 (SYSY, Göttingen, Germany), mouse anti-MAP2 (Sigma-Aldrich, St. Louis, MO, USA), and mouse anti-PSD95 (NeuroMab, Davis, CA, USA). Secondary antibodies were: Cy™ 3 goat anti-rabbit IgG (H + L) (Dianova, Hamburg, Germany), Alexa Fluor® donkey anti-mouse 568 IgG (H + L) (Invitrogen, Carlsbad, CA, USA), Alexa Fluor® 488 donkey anti-mouse IgG (H + L) (Invitrogen, Carlsbad, CA, USA), Alexa Fluor® 488 donkey anti-rabbit IgG (H + L) (Invitrogen, Carlsbad, CA, USA), Cy™ 3 donkey anti-guinea pig IgG (H + L) (Dianova, Hamburg, Germany), donkey anti-mouse 647 IgG (H + L) (Invitrogen, Carlsbad, CA, USA), Alexa Fluor® 488 donkey anti-rat IgG (H + L) (Invitrogen, Carlsbad, CA, USA), peroxidase-coupled AffiniPure donkey anti-rabbit IgG (H + L) (Jackson ImmunoResearch, Cambridgeshire, UK), and peroxidase-coupled AffiniPure donkey anti-mouse IgG (H + L) (Jackson ImmunoResearch, Cambridgeshire, UK). Drugs were used